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1.
PLoS Biol ; 18(3): e3000614, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32126082

RESUMO

The reproductive hormones that trigger oocyte meiotic maturation and release from the ovary vary greatly between animal species. Identification of receptors for these maturation-inducing hormones (MIHs) and understanding how they initiate the largely conserved maturation process remain important challenges. In hydrozoan cnidarians including the jellyfish Clytia hemisphaerica, MIH comprises neuropeptides released from somatic cells of the gonad. We identified the receptor (MIHR) for these MIH neuropeptides in Clytia using cell culture-based "deorphanization" of candidate oocyte-expressed G protein-coupled receptors (GPCRs). MIHR mutant jellyfish generated using CRISPR-Cas9 editing had severe defects in gamete development or in spawning both in males and females. Female gonads, or oocytes isolated from MIHR mutants, failed to respond to synthetic MIH. Treatment with the cAMP analogue Br-cAMP to mimic cAMP rise at maturation onset rescued meiotic maturation and spawning. Injection of inhibitory antibodies to the alpha subunit of the Gs heterodimeric protein (GαS) into wild-type oocytes phenocopied the MIHR mutants. These results provide the molecular links between MIH stimulation and meiotic maturation initiation in hydrozoan oocytes. Molecular phylogeny grouped Clytia MIHR with a subset of bilaterian neuropeptide receptors, including neuropeptide Y, gonadotropin inhibitory hormone (GnIH), pyroglutamylated RFamide, and luqin, all upstream regulators of sexual reproduction. This identification and functional characterization of a cnidarian peptide GPCR advances our understanding of oocyte maturation initiation and sheds light on the evolution of neuropeptide-hormone systems.


Assuntos
Hidrozoários/fisiologia , Neuropeptídeos/metabolismo , Oócitos/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas , AMP Cíclico/metabolismo , Feminino , Expressão Gênica , Hidrozoários/genética , Masculino , Mutação , Filogenia , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo
2.
Dev Biol ; 468(1-2): 59-79, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32976840

RESUMO

The cnidarian "planula" larva shows radial symmetry around a polarized, oral-aboral, body axis and comprises two epithelia cell layers, ectodermal and endodermal. This simple body plan is set up during gastrulation, a process which proceeds by a variety of modes amongst the diverse cnidarian species. In the hydrozoan laboratory model Clytia hemisphaerica, gastrulation involves a process termed unipolar cell ingression, in which the endoderm derives from mass ingression of individual cells via a process of epithelial-mesenchymal transition (EMT) around the future oral pole of an epithelial embryo. This contrasts markedly from the gastrulation mode in the anthozoan cnidarian Nematostella vectensis, in which endoderm formation primarily relies on cell sheet invagination. To understand the cellular basis of gastrulation in Clytia we have characterized in detail successive cell morphology changes during planula formation by Scanning and Transmission Electron Microscopy combined with confocal imaging. These changes successively accompany epithelialization of the blastoderm, EMT occurring in the oral domain through the bottle cell formation and ingression, cohesive migration and intercalation of ingressed cells with mesenchymal morphology, and their epithelialization to form the endoderm. From our data, we have reconstructed the cascade of morphogenetic events leading to the formation of planula larva. We also matched the domains of cell morphology changes to the expression of selected regulatory and marker genes expressed during gastrulation. We propose that cell ingression in Clytia not only provides the endoderm, but generates internal forces that shape the embryo in the course of gastrulation. These observations help build a more complete understanding of the cellular basis of morphogenesis and of the evolutionary plasticity of cnidarian gastrulation modes.


Assuntos
Padronização Corporal/fisiologia , Embrião não Mamífero/embriologia , Hidrozoários/embriologia , Animais , Larva
3.
PLoS Genet ; 10(9): e1004590, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25233086

RESUMO

We have used Digital Gene Expression analysis to identify, without bilaterian bias, regulators of cnidarian embryonic patterning. Transcriptome comparison between un-manipulated Clytia early gastrula embryos and ones in which the key polarity regulator Wnt3 was inhibited using morpholino antisense oligonucleotides (Wnt3-MO) identified a set of significantly over and under-expressed transcripts. These code for candidate Wnt signaling modulators, orthologs of other transcription factors, secreted and transmembrane proteins known as developmental regulators in bilaterian models or previously uncharacterized, and also many cnidarian-restricted proteins. Comparisons between embryos injected with morpholinos targeting Wnt3 and its receptor Fz1 defined four transcript classes showing remarkable correlation with spatiotemporal expression profiles. Class 1 and 3 transcripts tended to show sustained expression at "oral" and "aboral" poles respectively of the developing planula larva, class 2 transcripts in cells ingressing into the endodermal region during gastrulation, while class 4 gene expression was repressed at the early gastrula stage. The preferential effect of Fz1-MO on expression of class 2 and 4 transcripts can be attributed to Planar Cell Polarity (PCP) disruption, since it was closely matched by morpholino knockdown of the specific PCP protein Strabismus. We conclude that endoderm and post gastrula-specific gene expression is particularly sensitive to PCP disruption while Wnt-/ß-catenin signaling dominates gene regulation along the oral-aboral axis. Phenotype analysis using morpholinos targeting a subset of transcripts indicated developmental roles consistent with expression profiles for both conserved and cnidarian-restricted genes. Overall our unbiased screen allowed systematic identification of regionally expressed genes and provided functional support for a shared eumetazoan developmental regulatory gene set with both predicted and previously unexplored members, but also demonstrated that fundamental developmental processes including axial patterning and endoderm formation in cnidarians can involve newly evolved (or highly diverged) genes.


Assuntos
Polaridade Celular/genética , Cnidários/embriologia , Cnidários/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Transcriptoma/genética , Via de Sinalização Wnt/genética , Animais , Padronização Corporal/genética , Endoderma/embriologia , Feminino , Gástrula/embriologia , Gastrulação/genética , Larva/genética , Masculino , Proteínas de Membrana/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , beta Catenina/genética
4.
Sci Adv ; 7(48): eabh1683, 2021 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-34826233

RESUMO

We present an organism-wide, transcriptomic cell atlas of the hydrozoan medusa Clytia hemisphaerica and describe how its component cell types respond to perturbation. Using multiplexed single-cell RNA sequencing, in which individual animals were indexed and pooled from control and perturbation conditions into a single sequencing run, we avoid artifacts from batch effects and are able to discern shifts in cell state in response to organismal perturbations. This work serves as a foundation for future studies of development, function, and regeneration in a genetically tractable jellyfish species. Moreover, we introduce a powerful workflow for high-resolution, whole-animal, multiplexed single-cell genomics that is readily adaptable to other traditional or nontraditional model organisms.

5.
Elife ; 92020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32894220

RESUMO

Jellyfish, with their tetraradial symmetry, offer a novel paradigm for addressing patterning mechanisms during regeneration. Here we show that an interplay between mechanical forces, cell migration and proliferation allows jellyfish fragments to regain shape and functionality rapidly, notably by efficient restoration of the central feeding organ (manubrium). Fragmentation first triggers actomyosin-powered remodeling that restores body umbrella shape, causing radial smooth muscle fibers to converge around 'hubs' which serve as positional landmarks. Stabilization of these hubs, and associated expression of Wnt6, depends on the configuration of the adjoining muscle fiber 'spokes'. Stabilized hubs presage the site of the manubrium blastema, whose growth is Wnt/ß-catenin dependent and fueled by both cell proliferation and long-range cell recruitment. Manubrium morphogenesis is modulated by its connections with the gastrovascular canal system. We conclude that body patterning in regenerating jellyfish emerges mainly from local interactions, triggered and directed by the remodeling process.


Assuntos
Padronização Corporal/fisiologia , Hidrozoários/fisiologia , Regeneração/fisiologia , Animais , Movimento Celular , Hidrozoários/citologia , Hidrozoários/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt
6.
Biol Open ; 9(11)2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-32994186

RESUMO

The jellyfish species Clytia hemisphaerica (Cnidaria, Hydrozoa) has emerged as a new experimental model animal in the last decade. Favorable characteristics include a fully transparent body suitable for microscopy, daily gamete production and a relatively short life cycle. Furthermore, whole genome sequence assembly and efficient gene editing techniques using CRISPR/Cas9 have opened new possibilities for genetic studies. The quasi-immortal vegetatively-growing polyp colony stage provides a practical means to maintain mutant strains. In the context of developing Clytia as a genetic model, we report here an improved whole life cycle culture method including an aquarium tank system designed for culture of the tiny jellyfish form. We have compared different feeding regimes using Artemia larvae as food and demonstrate that the stage-dependent feeding control is the key for rapid and reliable medusa and polyp rearing. Metamorphosis of the planula larvae into a polyp colony can be induced efficiently using a new synthetic peptide. The optimized procedures detailed here make it practical to generate genetically modified Clytia strains and to maintain their whole life cycle in the laboratory.This article has an associated First Person interview with the two first authors of the paper.


Assuntos
Hidrozoários/crescimento & desenvolvimento , Hidrozoários/genética , Estágios do Ciclo de Vida/genética , Modelos Genéticos , Animais , Estudos de Associação Genética , Humanos , Larva , Metamorfose Biológica , Modelos Animais
7.
Nat Ecol Evol ; 3(5): 801-810, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30858591

RESUMO

Jellyfish (medusae) are a distinctive life-cycle stage of medusozoan cnidarians. They are major marine predators, with integrated neurosensory, muscular and organ systems. The genetic foundations of this complex form are largely unknown. We report the draft genome of the hydrozoan jellyfish Clytia hemisphaerica and use multiple transcriptomes to determine gene use across life-cycle stages. Medusa, planula larva and polyp are each characterized by distinct transcriptome signatures reflecting abrupt life-cycle transitions and all deploy a mixture of phylogenetically old and new genes. Medusa-specific transcription factors, including many with bilaterian orthologues, associate with diverse neurosensory structures. Compared to Clytia, the polyp-only hydrozoan Hydra has lost many of the medusa-expressed transcription factors, despite similar overall rates of gene content evolution and sequence evolution. Absence of expression and gene loss among Clytia orthologues of genes patterning the anthozoan aboral pole, secondary axis and endomesoderm support simplification of planulae and polyps in Hydrozoa, including loss of bilateral symmetry. Consequently, although the polyp and planula are generally considered the ancestral cnidarian forms, in Clytia the medusa maximally deploys the ancestral cnidarian-bilaterian transcription factor gene complement.


Assuntos
Hidrozoários , Animais , Evolução Molecular , Genoma
8.
Mol Biol Cell ; 14(3): 1125-37, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12631728

RESUMO

We have used complementary biochemical and in vivo approaches to study the compartmentalization of M phase-promoting factor (MPF) in prophase Xenopus eggs and oocytes. We first examined the distribution of MPF (Cdc2/CyclinB2) and membranous organelles in high-speed extracts of Xenopus eggs made during mitotic prophase. These extracts were found to lack mitochondria, Golgi membranes, and most endoplasmic reticulum (ER) but to contain the bulk of the pre-MPF pool. This pre-MPF could be pelleted by further centrifugation along with components necessary to activate it. On activation, Cdc2/CyclinB2 moved into the soluble fraction. Electron microscopy and Western blot analysis showed that the pre-MPF pellet contained a specific ER subdomain comprising "annulate lamellae" (AL): stacked ER membranes highly enriched in nuclear pores. Colocalization of pre-MPF with AL was demonstrated by anti-CyclinB2 immunofluorescence in prophase oocytes, in which AL are positioned close to the vegetal surface. Green fluorescent protein-CyclinB2 expressed in oocytes also localized at AL. These data suggest that inactive MPF associates with nuclear envelope components just before activation. This association may explain why nuclei and centrosomes stimulate MPF activation and provide a mechanism for targeting of MPF to some of its key substrates.


Assuntos
Fator Promotor de Maturação/metabolismo , Mitose/fisiologia , Oócitos/química , Oócitos/fisiologia , Oócitos/ultraestrutura , Xenopus laevis/fisiologia , Animais , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fracionamento Celular , Membrana Celular/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Retículo Endoplasmático/metabolismo , Feminino , Membrana Nuclear/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Extratos de Tecidos/química , Fosfatases cdc25/metabolismo
9.
Mol Biol Cell ; 22(12): 2042-53, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21508313

RESUMO

The atypical protein kinase C (aPKC) is part of the conserved aPKC/PAR6/PAR3 protein complex, which regulates many cell polarity events, including the formation of a primary cilium at the apical surface of epithelial cells. Cilia are highly organized, conserved, microtubule-based structures involved in motility, sensory processes, signaling, and cell polarity. We examined the distribution and function of aPKC in the sea urchin embryo, which forms a swimming blastula covered with motile cilia. We found that in the early embryo aPKC is uniformly cortical and becomes excluded from the vegetal pole during unequal cleavages at the 8- to 64-cell stages. During the blastula and gastrula stages the kinase localizes at the base of cilia, forming a ring at the transition zone between the basal body and the elongating axoneme. A dose-dependent and reversible inhibition of aPKC results in mislocalization of the kinase, defective ciliogenesis, and lack of swimming. Thus, as in the primary cilium of differentiated mammalian cells, aPKC controls the growth of motile cilia in invertebrate embryos. We suggest that aPKC might function to phosphorylate kinesin and so activate the transport of intraflagellar vesicles.


Assuntos
Proteína Quinase C/metabolismo , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/metabolismo , Animais , Polaridade Celular , Cílios/metabolismo , Embrião não Mamífero/metabolismo , Células Epiteliais/metabolismo , Indóis/farmacologia , Cinesinas/metabolismo , Maleimidas/farmacologia , Microtúbulos/metabolismo , Fosforilação , Isoformas de Proteínas/metabolismo , Proteína Quinase C/antagonistas & inibidores
10.
PLoS One ; 5(11): e13994, 2010 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-21103375

RESUMO

Poc1 (Protein of Centriole 1) proteins are highly conserved WD40 domain-containing centriole components, well characterized in the alga Chlamydomonas, the ciliated protazoan Tetrahymena, the insect Drosophila and in vertebrate cells including Xenopus and zebrafish embryos. Functions and localizations related to the centriole and ciliary axoneme have been demonstrated for Poc1 in a range of species. The vertebrate Poc1 protein has also been reported to show an additional association with mitochondria, including enrichment in the specialized "germ plasm" region of Xenopus oocytes. We have identified and characterized a highly conserved Poc1 protein in the cnidarian Clytia hemisphaerica. Clytia Poc1 mRNA was found to be strongly expressed in eggs and early embryos, showing a punctate perinuclear localization in young oocytes. Fluorescence-tagged Poc1 proteins expressed in developing embryos showed strong localization to centrioles, including basal bodies. Anti-human Poc1 antibodies decorated mitochondria in Clytia, as reported in human cells, but failed to recognise endogenous or fluorescent-tagged Clytia Poc1. Injection of specific morpholino oligonucleotides into Clytia eggs prior to fertilization to repress Poc1 mRNA translation interfered with cell division from the blastula stage, likely corresponding to when neosynthesis normally takes over from maternally supplied protein. Cell cycle lengthening and arrest were observed, phenotypes consistent with an impaired centriolar biogenesis or function. The specificity of the defects could be demonstrated by injection of synthetic Poc1 mRNA, which restored normal development. We conclude that in Clytia embryos, Poc1 has an essentially centriolar localization and function.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Embrião não Mamífero/metabolismo , Hidrozoários/metabolismo , Animais , Western Blotting , Proteínas de Ciclo Celular/classificação , Proteínas de Ciclo Celular/genética , DNA Complementar/química , DNA Complementar/genética , Embrião não Mamífero/embriologia , Feminino , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Biblioteca Gênica , Hidrozoários/embriologia , Hidrozoários/genética , Hibridização In Situ , Microscopia Confocal , Dados de Sequência Molecular , Oócitos/metabolismo , Filogenia , Análise de Sequência de DNA
11.
Curr Biol ; 19(4): 305-11, 2009 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-19230670

RESUMO

The kinase Mos, which activates intracellularly the MAP kinase pathway, is a key regulator of animal oocyte meiotic maturation. In vertebrate and echinoderm models, Mos RNA translation upon oocyte hormonal stimulation mediates "cytostatic" arrest of the egg after meiosis, as well as diverse earlier events [1-5]. Our phylogenetic survey has revealed that MOS genes are conserved in cnidarians and ctenophores, but not found outside the metazoa or in sponges. We demonstrated MAP kinase-mediated cytostatic activity for Mos orthologs from Pleurobrachia (ctenophore) and Clytia (cnidarian) by RNA injection into Xenopus blastomeres. Analyses of endogenous Mos in Clytia with morpholino antisense oligonucleotides and pharmacological inhibition demonstrated that Mos/MAP kinase function in postmeiotic arrest is conserved. They also revealed additional roles in spindle formation and positioning, strongly reminiscent of observations in starfish, mouse, and Xenopus. Unusually, cnidarians were found to possess multiple Mos paralogs. In Clytia, one of two maternally expressed paralogs accounted for the majority MAP kinase activation during maturation, whereas the other may be subject to differential translational regulation and have additional roles. Our findings indicate that Mos appeared early during animal evolution as an oocyte-expressed kinase and functioned ancestrally in regulating core specializations of female meiosis.


Assuntos
Cnidários/enzimologia , Ctenóforos/enzimologia , Oócitos/enzimologia , Proteínas Proto-Oncogênicas c-mos/metabolismo , Animais , Evolução Biológica , Cnidários/citologia , Ctenóforos/citologia , Feminino , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Meiose/fisiologia , Camundongos , Microtúbulos/metabolismo , Dados de Sequência Molecular , Oócitos/citologia , Filogenia , Proteínas Proto-Oncogênicas c-mos/classificação , Proteínas Proto-Oncogênicas c-mos/genética
12.
Dev Genes Evol ; 216(11): 709-20, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17021866

RESUMO

We have characterised the expression of four genes coding for Forkhead box-containing ('Fox') transcription factors identified from the hydrozoan (Leptomedusa) Clytia hemisphaerica. Phylogenetic analyses including all available non-bilaterian Fox sequences placed these genes in subfamilies B, Q2 (two genes) and O, and indicated that at least 17 Fox subfamilies were present in the common cnidarian/bilaterian ancestor, with multiple subsequent losses in cnidarian lineages. Chordate FoxB and FoxQ2A subfamily genes show polarised expression in early embryos. Correspondingly, Clytia CheFoxB expression was localised around the gastrulation site (future oral pole) at blastula and gastrula stages, with CheFoxQ2a expressed in a complementary aboral domain, maintained through larval development. Distinct later expression domains were observed for CheFoxB in the larval endoderm region, and in the statocyst, gonad and tentacle bulb of the medusa. A second Clytia FoxQ2 gene, CheFoxQ2b, not expressed in the embryo, larva or polyp, was detected uniquely in the gonads of the medusa. In contrast, CheFoxO, whose sequence indicates regulation by the PI3-Kinase/PKB signalling pathway consistent with known roles in bilaterian developmental regulation, was detected throughout the Clytia life cycle. CheFoxO expression was enhanced in regions associated with growth control including larval poles, gonad and the margin of the medusa bell. These results support the idea that an early embryonic patterning system involving FoxB and FoxQ2 family genes has been evolutionary conserved and indicate that Fox family genes have also acquired distinct roles during other phases of the hydrozoan life cycle.


Assuntos
Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Hidrozoários/genética , Sequência de Aminoácidos , Animais , Padronização Corporal , DNA Complementar , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos
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